Stem cell therapies have the potential to dramatically change the treatment of a number of diseases such as Parkinson's, Alzheimer's, spinal cord injury, diabetes, ischemia stroke and heart disease. Although tremendous achievements and rapid developments have been made in the past decade for cell-based therapies of a number of disease states, the potential that stem cells offer remains to be better understood. One of the concerns with stem cell therapies is the risk that transplanted stem cells could form tumors and become cancerous if cell division continues uncontrollably. Critical insights can be achieved by observing their fate in vivo overtime using noninvasive imaging techniques.
An emerging technology that allows for visualization of interactions between molecular probes and biological targets is molecular imaging, which can be divided into two general categories: (1) the direct labeling method, and (2) the reporter gene approach. The former involves using an imaging-detectable probe that can be loaded into cells and would remain intracellular during tracking. This method does not involve extensive manipulation of cells and therefore is preferred for clinical implementation. It has two inherent limitations: labels may be diluted upon cell division, making the cells eventually invisible; and labels may efflux from cells or may degrade over time. Examples of contrast agents are FDG for PET, 111In-oxine for SPECT and iron nanoparticles (SPIOs) for MRI, which have significant limitations. (Zhang, et al. 2008 Current Pharma. Design 14 (36): 3835-3853; Zhou, et al. 2006 JACC 48 (10): 2094-2106.)
The reporter gene approach is used mostly for preclinical studies. (Narsinh, et al. 2009 Molecular Imaging of Human Embryonic Stem Cells. Ch 2 p 13-32; in Methods in Molecular Biology, Viral Applications of Green Fluorescent Protein, Vol. 515. Barry W Hicks (Ed) Humana Press.) This approach involves inserting a reporter gene(s) into the stem cell that can then be tracked upon administration of a reporter probe. Reporter genes are useful for assessing the longer-term survival of the implanted cells because the reporter will be expressed as long as the cells are alive. Major disadvantages of the reporter gene approach include: (i) stable transfection (lentiviral or retroviral) involves extensive molecular manipulation of the cells under study and runs the risk of insertional mutagenesis; (ii) immune reactions may be induced; (iii) uncertainties regarding the robustness of the signal remain, as the detected signal could reflect magnitude of transgene expression instead of cell survival; (iv) gene modification adds additional cost; and (v) regulatory roadblocks. (Frangioni, et al. 2004 Circulation 110: 3378-3384.)
To date, most stem cell tracking studies have used direct in vitro cell labeling with SPIO followed by in vivo MRI (e.g., neural stem cells were tracked in vivo for up to 18 weeks. (Guzman, et al. 2007 PNAS (USA) 104, 11915-11920.) Despite the significant advantages of MRI (25-100 μm resolution, excellent anatomical and functional data), it has been found that iron-derived signals can persist in organs (e.g., myocardium) and can be detected by MRI long after the cells have been destroyed, thus generating false-positive signals. (Wang, et al. 2008 British J. Radiology 81: 987-988; Terrovitis, et al. 2008 Circulation 117: 1555-62.) SPIO labeling was also found to affect SC migration. (Schafer, et al. 2009 Cytotherapy 11 (1): 68-78.) Another significant clinical problem common to all MRI methods is that certain implantable devices such as pacemakers and defibrillators, are currently contraindications to MRI.
Nuclear imaging techniques, single-photon emission computed tomography (SPECT) and positron emission tomography (PET), offer high sensitivity (10−11M-10-12M tracer) deep in tissue. Specialized systems of both PET and SPECT allow small animal imaging with much improved spatial resolution (1-2 mm). SPECT imaging provides 3D information and can be applied both in small animals as well as in humans. Although the sensitivity of PET is 1-2 orders of magnitude better than SPECT, SPECT is less expensive, more widely available, and allows multispectral detection and uses isotopes with longer half-lives. (Stodilka, et al. 2006 Phys. Med. Biol. 51: 2619-2632.)
Optical imaging is a relatively new imaging modality that offers real-time, non-radioactive, and depending on the technique, high-resolution imaging of fluorochromes embedded in diseased tissues, e.g., cellular resolution is possible using microscopy techniques. Far red (FR) and near infrared (NIR)(650-900 nm wavelengths) fluorescence-based imaging is of particular interest for noninvasive in vivo imaging because of the relatively low tissue absorption, scatter, and minimal autofluorescence of FR/NIR light. The sensitivity of this modality is comparable to nuclear techniques approaching a few thousand cells and the acquisition time is quite fast, obtaining images in seconds in most cases. (Zhang, et al. 2005 Bioconjugate Chem. 16: 1232-1239.)
Current radioisotope and optical probes for stem cell tracking have limitations. Direct cell labeling has previously been used for early tracking of transplanted stem cells into the myocardium in clinical trials. The most widely used reporter gene for nuclear imaging is HSV-tk based PET imaging using [18F]-FHBG as the reporter probe. HSV-tk has the additional property of serving as a suicide gene upon administration of ganciclovir, thereby allowing selective ablation of stem cell misbehavior. However, this approach suffers from many of the same limitations previously mentioned.
Direct radiolabeling of cells has traditionally been accomplished with the incorporation of lipophilic chelates: 111In-oxine, 111In-tropolone, or 99mTc-HMPAO (hexamethylpropylene amine oxime). For stem cell tracking, the short radiological half-life of 99mTc (6.02 h) is not particularly useful for longer term imaging studies using the direct radiolabeling approach. MSCs and endothelial progenitor cells (EPCs) labeled with 111In-oxine or 99mTc-HMPAO have been monitored in vivo using SPECT in animal studies (e.g., radiolabeling of progenitor cells with 111In is feasible for monitoring myocardial homing and biodistribution in rats over 24-48 h. (Brenner, et al. 2004 J. Nucl. Med. 45 (3): 512-518.)
One problem reported with cells labeled with 111In-oxine or 111In-tropolone is the potential for accumulation of the isotope in the nucleus, resulting in radiotoxicity and limiting the amount of label possible per cell. This is due to the short range of the emitted low-energy B-particles, causing severe chromosomal aberration. (ten Berge, et al. 1983 J. Nucl. Med. 24: 615-620.) Evidence from a number of studies has shown that radiation damage from Auger-electron emitters such as 111In can be reduced 85-fold if the nuclide is confined to the cytoplasm rather than the nucleus. (Lambert, et al. 1996 Nucl. Med. & Biol. 23:417-427.) If the nuclide is restricted to the cell membrane, radiation damage can be reduced 120-fold. (Hofer, K H. 1984 Microdosimetry of labeled cells. In Blood Cells in Nuclear Medicine, Part II (Edited by Fueger, GF), Martinus Nijhoff Publishers, Boston; 224-243.)
The cytotoxic effects of 111In on human stem cells have also been reported to be time-dependent. (Gholamrezanezhad, et al. 2009 Nucl. Med. Commun. 30 (3): 210-216.) Detection at the single cell level remains a formidable challenge for radionuclide probes as the ability to concentrate radioactive agents in stem cells has yet to be achieved. (Frangioni, et al. 2004 Circulation 110: 3378-3384.)
Another important limitation that has been reported with 111In-oxine and 111In-tropolone is the leaking of the label from the stem cell resulting in false positive signals and also high uptake in liver and kidneys. (Brenner, et al. 2004 J. Nucl. Med. 45 (3): 512-518; Zhou, et al. 2005 J. Nucl. Med. 46 (5): 816-822.) This is because binding of these compounds to intracellular structures is reversible.
SPECT has been used to track 111In-labeled transplanted progenitor cells in murine, porcine and canine models of myocardial infaction until 14 days. (Aicher, et al. 2003 Circulation 107, 2134-9; Chin, et al. 2003 Nucl. Med. Commun. 24, 1149-54; Wisenberg, et al. 2009 J. Cardiovascular Magnetic Reson. 11: 11-26.) Jin et al. demonstrated that stem cells can be radiolabeled with indium up to 0.14 Bq/cell without affecting viability and function, and detected as few as 3600 cells so radiolabeled by SPECT in a phantom study. (Jin, et al. 2005 Phys. Med. Biol. 50: 4445-4455.)
A dual modality cell labeling probe reported is 125I-PKH95. (Slezak, et al. 1991 Nature 352:261-262; Ford, et al. 1996 J. Surgical Res. 62 (1): 23-28.) The 125I-PKH95 compound contains an iodine atom, a visible fluorescent head group (em=570 nm) and two long alkyl tails that enable it to stably embed into cell membranes irreversibly. It was labeled by exchange with 125I, but with low specific activity (15-40 Ci/mmol). (Gray, et al. 1991 J. Nucl. Med. 32: 1092.)
The use of an iodine isotope presents practical issues. First, exchange labeling is not efficient in producing a high specific radioactivity agent. High specific-activity is necessary to minimize the total number of dye molecules associated with each cell to diminish detrimental effects on cellular integrity. Second, it is challenging to conform to a simple kit format that can be easily radiolabeled on site.
Thus, new dual- or multi-modality probes for stem cell tracking are needed. Labeling stem cells with new dual- or multi-modality probes and tracking them via noninvasive imaging techniques may hold the key to addressing critical issues associated with successful development of stem cell therapies.